Abstract:

The present invention provides stereoisomers and stereoisomeric mixtures
of 3-aminocarbonyl-bicycloheptene-2,4-pyrimidinediamine compounds having
antiproliferative activity, compositions comprising the compounds and
methods of using the compounds to inhibit cellular proliferation and to
treat proliferate diseases such as tumorigenic cancers.

Claims:

1. A pharmaceutically acceptable salt of a compound according to
structural formula (I): ##STR00073## or an N-oxide thereof, that is
enriched in the diastereomer of structural formula (Ia): ##STR00074##
wherein:each R1 is independently selected from the group consisting
of hydrogen, lower alkyl, --(CH2)n--OH, --ORa,
--O(CH2)n--Ra, --O(CH2)nRb, --C(O)ORa,
halo, --CF3 and --OCF3;each R2 is independently selected
from the group consisting of hydrogen, lower alkyl --ORa,
--O(CH2)n--Ra, --O(CH2)n--Rb,
--NHC(O)Ra, halo, --CF3, --OCF3, ##STR00075## each R3
is independently selected from the group consisting of hydrogen, lower
alkyl, --(CH2)n--OH, --ORa, --O(CH2)n--Ra,
--O(CH2)n--Rb, halo, --CF3, --OCF3, ##STR00076##
each R4 is independently selected from the group consisting of
hydrogen, lower alkyl, arylalkyl, --ORa, --NRcRc,
--C(O)Ra, --C(O)ORa and --C(O)NRcRc;R5 is
hydrogen, halo, fluoro, --CN, --NO2, CO2Ra or
--CF3;each n is independently an integer from 1 to 3;each Ra is
independently selected from the group consisting of hydrogen, lower alkyl
and lower cycloalkyl;each Rb is independently selected from the
group consisting of --ORa, --CF3, --OCF3,
--NRcRc, --C(O)Ra, --C(O)ORa, --C(O)NRcRc
and --C(O)NRaRd;each Rc is independently selected from the
group consisting of hydrogen and lower alkyl, or, alternatively, two
Rc substituents may be taken together with the nitrogen atom to
which they are bonded to form a 5-7 membered saturated ring which
optionally includes 1-2 additional heteroatomic groups selected from O,
NRa, NRa--C(O)Ra, NRa--C(O)ORa and
NRa--C(O)NRa; andeach Rd is independently lower
mono-hydroxyalkyl or lower di-hydroxyalkylwherein the pharmaceutically
acceptable salt is an acid addition salt.

2. The pharmaceutically acceptable salt of claim 1 in which the compound
according to structural formula (I) is a (2-exo-3-exo) cis racemate.

3. The pharmaceutically acceptable salt of claim 1 which contains about
60% or more of the diastereomer of structural formula (Ia).

4. The pharmaceutically acceptable salt of claim 1 which contains about
90% or more of the diastereomer of structural formula (Ia).

5. The pharmaceutically acceptable salt of claim 1 which contains about
99% or more of the diastereomer of structural formula (Ia).

6. The pharmaceutically acceptable salt of claim 1 in which R5 is
fluoro.

7. (canceled)

8. (canceled)

9. (canceled)

10. (canceled)

11. (canceled)

12. (canceled)

13. (canceled)

14. (canceled)

15. (canceled)

16. The pharmaceutically acceptable salt of claim 6 in which R1 is
hydrogen; R2 is selected from the group consisting of hydrogen,
##STR00077## and R3 is selected from the group consisting of
hydrogen, lower alkyl, halo, --CF3, ##STR00078##

17. The pharmaceutically acceptable salt of claim 16 in which R3 is
selected from the group consisting of hydrogen, methyl, chloro,
--CF3, ##STR00079## and R4 is methyl, --CORa or
--CO(O)Ra where Ra is methyl or ethyl.

18. The pharmaceutically acceptable salt of claim 16 in which R2 is
selected from the group consisting of hydrogen, ##STR00080## and R3
is selected from the group consisting of hydrogen, lower alkyl, halo,
--CF3, ##STR00081##

19. The pharmaceutically acceptable salt of claim 18 in which R3 is
selected from the group consisting of hydrogen, methyl, chloro,
--CF3, ##STR00082## and R4 is methyl, --CORa or
--CO(O)Ra wherein Ra is methyl or ethyl.

20. (canceled)

21. (canceled)

22. (canceled)

23. (canceled)

24. The pharmaceutically acceptable salt of claim 19 in which R2 is
##STR00083## R4 is methyl; and R3 is selected from the group
consisting of hydrogen, methyl, chloro and --CF.sub.3.

25. The pharmaceutically acceptable salt of claim 24 in which R3 is
methyl.

26. (canceled)

27. (canceled)

28. A composition comprising a pharmaceutically acceptable salt according
to claim 1 and a pharmaceutically acceptable carrier, excipient and/or
diluent.

[0002]The present disclosure relates to stereoisomerically enriched
compositions of
4N-(3-aminocarbonylbicyclo[2.2.1]hept-5-en-2-yl)-N2-substituted
phenyl-2,4-pyrimidinediamine compounds that exhibit antiproliferative
activity, prodrugs of the compounds, intermediates and methods of
synthesis for making the compounds and/or prodrugs, pharmaceutical
compositions comprising the compounds and/or prodrugs and the use of the
compounds and/or prodrugs in a variety of contexts, including, for
example, the treatment of proliferative disorders, such as tumors and
cancers.

3. BACKGROUND

[0003]Cancer is a group of varied diseases characterized by uncontrolled
growth and spread of abnormal cells. Generally, all types of cancers
involve some abnormality in the control of cell growth and division. The
pathways regulating cell division and/or cellular communication become
altered in cancer cells such that the effects of these regulatory
mechanisms in controlling and limiting cell growth fails or is bypassed.
Through successive rounds of mutation and natural selection, a group of
abnormal cells, generally originating from a single mutant cell,
accumulates additional mutations that provide selective growth advantage
over other cells, and thus evolves into a cell type that predominates in
the cell mass. This process of mutation and natural selection is enhanced
by genetic instability displayed by many types of cancer cells, an
instability which is gained either from somatic mutations or by
inheritance from the germ line. The enhanced mutability of cancerous
cells increases the probability of their progression towards formation of
malignant cells. As the cancer cells further evolve, some become locally
invasive and then mestasize to colonize tissues other than the cancer
cell's tissue of origin. This property along with the heterogeneity of
the tumor cell population makes cancer a particularly difficult disease
to treat and eradicate.

[0004]Traditional cancer treatments take advantage of the higher
proliferative capacity of cancer cells and their increased sensitivity to
DNA damage. Ionizing radiation, including γ-rays and x-rays, and
cytotoxic agents, such as bleomycin, cis-platin, vinblastine,
cyclophosphamide, 5'-fluorouracil, and methotrexate rely upon a
generalized damage to DNA and destabilization of chromosomal structure
which eventually lead to destruction of cancer cells. These treatments
are particularly effective for those types of cancers that have defects
in cell cycle checkpoint, which limits the ability of these cells to
repair damaged DNA before undergoing cell division. The non-selective
nature of these treatments, however, often results in severe and
debilitating side effects. The systemic use of these drugs may result in
damage to normally healthy organs and tissues, and compromise the
long-term health of the patient.

[0005]Although more selective chemotherapeutic treatments have been
developed based on knowledge of how cancer cells develop, for example,
the anti-estrogen compound tamoxifen, the effectiveness of all
chemotherapeutic treatments are subject to development of resistance to
the drugs. In particular, the increased expression of cell membrane bound
transporters, such as MdrI, produces a multidrug resistance phenotype
characterized by increased efflux of drugs from the cell. These types of
adaptation by cancer cells severely limit the effectiveness of certain
classes of chemotherapeutic agents. Consequently, identification of other
chemotherapeutic agents, particularly active stereoisomers and/or
stereoisomeric mixtures is critical for establishing therapies effective
for attacking the heterogeneous nature of proliferative disease and for
overcoming any resistance that may develop over the course of therapy
with other compounds. Moreover, use of combinations of chemotherapeutic
agents, including different stereoisomers and/or stereoisomeric mixtures
of a particular chemotherapeutic agent, which may have differing
properties and cellular targets, increases the effectiveness of
chemotherapy and limits the generation of drug resistance.

4. SUMMARY

[0006]In one aspect,
4N-(3-aminocarbonylbicyclo[2.2.1]hept-5-en-2-yl)-2N-substituted
phenyl-2,4-pyrimidinediamine compounds enriched in specified
diastereomers are provided that exhibit antiproliferative activity
against a variety of different types of tumor cells. In some embodiments,
compounds according to structural formula (I) are provided:

##STR00001##

[0007]including prodrugs, salts, hydrates, solvates and N-oxides thereof,
that are enriched in the corresponding diastereomer of structural formula
(Ia), designated the (1R,2R,3S,4S) diastereomer:

[0012]each R4 is independently selected from the group consisting of
hydrogen, lower alkyl, arylalkyl, --ORa, --NRcRc,
--C(O)Ra, --C(O)ORa and --C(O)NRcRc; [0013]R5 is
hydrogen, halo, fluoro, --CN, --NO2, --C(O)ORa or --CF3;
[0014]each n is independently an integer from 1 to 3; [0015]each Ra
is independently selected from the group consisting of hydrogen, lower
alkyl and lower cycloalkyl; [0016]each Rb is independently selected
from the group consisting of --ORa, --CF3, --OCF3,
--NRcRc, --C(O)Ra, --C(O)ORa, --C(O)NRcRc
and --C(O)NRaRd; [0017]each Rc is independently selected
from the group consisting of hydrogen and lower alkyl, or, alternatively,
two Rc substituents may be taken together with the nitrogen atom to
which they are bonded to form a 4-9 membered saturated ring which
optionally includes 1-2 additional heteroatomic groups selected from O,
NRa, NRa--C(O)Ra, NRa--C(O)ORa and
NRa--C(O)NRa; and [0018]each Rd is independently lower
mono-hydroxyalkyl or lower di-hydroxyalkyl.

[0019]In some embodiments, the compound of structural formula (I) is a
racemic mixture of (2-exo-3-exo) cis isomers according to structural
formula (IIa):

[0021]In some embodiments, the compound is a stereoisomerically enriched
diastereomer according to structural formula (Ia), supra, including
prodrugs, salts, hydrates, solvates and N-oxides thereof, that is
substantially free of its enantiomer and any other diastereomer thereof.

[0022]In still another aspect, prodrugs of the stereoisomerically enriched
compounds are provided. Such prodrugs may be active in their prodrug
form, or may be inactive until converted under physiological or other
conditions of use to an active drug form. In the prodrugs, one or more
functional groups of the stereoisomerically enriched compounds are
included in promoieties that cleave from the molecule under the
conditions of use, typically by way of hydrolysis, enzymatic cleavage or
some other cleavage mechanism, to yield the functional groups. For
example, primary or secondary amino groups may be included in an amide
promoiety that cleaves under conditions of use to generate the primary or
secondary amino group. Thus, the prodrugs include special types of
protecting groups, termed "progroups," masking one or more functional
groups of the compounds that cleave under the conditions of use to yield
an active drug compound. Functional groups within the stereoisomerically
enriched compounds that may be masked with progroups for inclusion in a
promoiety include, but are not limited to, amines (primary and
secondary), hydroxyls, sulfanyls (thiols), carboxyls, carbonyls, etc.
Myriad progroups suitable for masking such functional groups to yield
promoieties that are cleavable under the desired conditions of use are
known in the art. All of these progroups, alone or in combination, may be
included in the prodrugs. Specific examples of promoieties that yield
primary or secondary amine groups that can be included in the prodrugs
include, but are not limited to amides, carbamates, imines, ureas,
phosphenyls, phosphoryls and sulfenyls. Specific examples of promoieties
that yield sulfanyl groups that can be included in the prodrugs include,
but are not limited to, thioethers, for example S-methyl derivatives
(monothio, dithio, oxythio, aminothio acetals), silyl thioethers,
thioesters, thiocarbonates, thiocarbamates, asymmetrical disulfides, etc.
Specific examples of promoieties that cleave to yield hydroxyl groups
that can be included in the prodrugs include, but are not limited to,
sulfonates, esters and carbonates. Specific examples of promoieties that
yield carboxyl groups that can be included in the prodrugs include, but
are not limited to, esters (including silyl esters, oxamic acid esters
and thioesters), amides and hydrazides.

[0023]In still another aspect, compositions comprising one or more
stereoisomerically enriched compounds are provided. The compositions
generally comprise the compound(s), and/or prodrugs, salts, hydrates,
solvates and/or N-oxides thereof, and an appropriate carrier, excipient
and/or diluent. The exact nature of the carrier, excipient and/or diluent
will depend upon the desired use for the composition, and may range from
being suitable or acceptable for in vitro uses, to being suitable or
acceptable for veterinary uses, to being suitable or acceptable for use
in humans.

[0024]The stereoisomerically enriched compounds described herein are
potent inhibitors of proliferation abnormal cells, such as tumor cells,
in in vitro assays. Thus, in still another aspect, methods of inhibiting
proliferation of abnormal cells, and in particular tumor cells, are
provided. The methods generally involve contacting an abnormal cell, such
as a tumor cell, with an amount of one or more stereoisomerically
enriched compounds described herein, and/or prodrugs, salts, hydrates,
solvates and/or N-oxides thereof, effective to inhibit proliferation of
the cell. The cells can be contacted with the compound per se, or the
compound can be formulated into a composition. The methods may be
practiced in in vitro contexts, or in in vivo contexts as a therapeutic
approach towards the treatment or prevention of proliferative disorders,
such as tumorigenic cancers.

[0025]In still another aspect, methods of treating proliferative disorders
are provided. The methods may be practiced in animals in veterinary
contexts or in humans. The methods generally involve administering to an
animal or human subject an amount of one or more stereoisomerically
enriched compounds described herein, and/or prodrugs, salts, hydrates,
solvates and/or N-oxides thereof, effective to treat or prevent the
proliferative disorder. The compound(s) per se can be administered to the
subject, or the compound(s) can be administered in the form of a
composition. Proliferative disorders that can be treated according to the
methods include, but are not limited to, tumorigenic cancers.

[0026]The stereoisomerically enriched compounds described herein are
potent inhibitors of Aurora kinases. Aurora kinases are a family of
enzymes known to be key regulators of cell division. Elevated levels of
Aurora kinases have been found in several types of human cancer cells,
such as breast, colon, renal, cervical, neuroblastomer, melanoma,
lymphoma, pancreatic, prostate and other solid tumors (see, e.g.,
Bischott et al., 1998, EMBO J. 17:3052-3065; Geopfert & Brinkley, 2000,
Curr. Top. Dev. Biol. 49:331-342; Sakakura et al., 2001, Br. J. Cancer
84:824-831), and overexpression of Aurora kinases has been shown to
result in cell transformation, a process by which normal cells become
cancers. Although not intending to be bound by any particular theory of
operation, it is believed that the stereoisomerically enriched compounds
described herein, as well as the active prodrugs, salts, hydrates,
solvates and/or N-oxides thereof, exert their antiproliferative activity
by inhibiting one or more Aurora kinases.

[0027]Thus, in yet another aspect, methods of inhibiting an activity of an
Aurora kinase are provided. The methods generally involve contacting an
Aurora kinase with an amount of one or more stereoisomerically enriched
compounds described herein, and/or active prodrugs, salts, hydrates,
solvates and/or N-oxides thereof, effective to inhibit its activity. The
methods can be practiced in in vitro contexts with purified or partially
purified Aurora kinase enzymes (e.g., with extracts of cells expressing
an Aurora kinase), in in vitro contexts with intact cells expressing an
Aurora kinase, or in in vivo contexts to inhibit an Aurora
kinase-mediated process (for example cellular mitotis) and/or as a
therapeutic approach towards the treatment or prevention of diseases or
disorders that are mediated, at least in part, by Aurora kinase activity.

[0028]In still another aspect, methods of treating or preventing Aurora
kinase-mediated diseases or disorders are provided. The methods generally
involve administering to an animal or human subject an amount of one or
more stereoisomerically enriched compounds described herein, and/or
active prodrugs, salts, hydrates, solvates and/or N-oxides thereof,
effective to treat or prevent the Aurora kinase-mediated disease or
disorder. Aurora kinase-mediated diseases and disorders include any
disease, disorder, or other deletarions condition in which a member of
the Aurora kinase family of enzymes plays a role. Specific examples of
such Aurora kinase-mediated diseases or disorders include, but are not
limited to, melanoma, leukemia, and solid tumor cancers, such as, for
example, colon, breast, gastric, ovarian, cervical, melanoma, renal,
prostate, lymphoma, neuroblastoma, pancreatic and bladder cancers.

[0029]Other aspects include, but are not limited to, intermediates and
methods useful for synthesizing the stereoisomerically enriched compounds
and prodrugs, as will be described in more detail herein below.

5. BRIEF DESCRIPTION OF THE DRAWINGS

[0030]FIGS. 1-4 illustrate the inhibitory effect of
(1R,2R,3S,4S)--N-4-(3-aminocarbonylbicyclo[2.2.1]hept-5-ene-2-yl)-5-fluor-
o-N2-[3-methyl-4-(4-methylpiperazin-1-yl)phenyl]-2,4-pyrimidinediamine bis
hydrogen chloride salt (compound 60a.2HCl) on the growth of various
different types of tumors in standard xenograft treatment and regression
models.

6. DETAILED DESCRIPTION

6.1 Definitions

[0031]As used herein, the following terms are intended to have the
following meanings:

[0032]"Alkyl" by itself or as part of another substituent refers to a
saturated or unsaturated branched, straight-chain or cyclic monovalent
hydrocarbon radical having the stated number of carbon atoms (i.e., C1-C6
means one to six carbon atoms) that is derived by the removal of one
hydrogen atom from a single carbon atom of a parent alkane, alkene or
alkyne. Typical alkyl groups include, but are not limited to, methyl;
ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl,
propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl,
prop-2-en-1-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl, prop-1-yn-1-yl,
prop-2-yn-1-yl, etc.; butyls such as butan-1-yl, butan-2-yl,
2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,
but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,
but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,
cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,
but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Where
specific levels of saturation are intended, the nomenclature "alkanyl,"
"alkenyl" and/or "alkynyl" is used, as defined below. "Lower alkyl"
refers to an alkyl group containing from 1 to 6 carbon atoms.

[0033]"Alkanyl" by itself or as part of another substituent refers to a
saturated branched, straight-chain or cyclic alkyl derived by the removal
of one hydrogen atom from a single carbon atom of a parent alkane.
Typical alkanyl groups include, but are not limited to, methanyl;
ethanyl; propanyls such as propan-1-yl, propan-2-yl (isopropyl),
cyclopropan-1-yl, etc.; butanyls such as butan-1-yl, butan-2-yl
(sec-butyl), 2-methyl-propan-1-yl (isobutyl), 2-methyl-propan-2-yl
(t-butyl), cyclobutan-1-yl, etc.; and the like.

[0034]"Alkenyl" by itself or as part of another substituent refers to an
unsaturated branched, straight-chain or cyclic alkyl having at least one
carbon-carbon double bond derived by the removal of one hydrogen atom
from a single carbon atom of a parent alkene. The group may be in either
the cis or trans conformation about the double bond(s). Typical alkenyl
groups include, but are not limited to, ethenyl; propenyls such as
prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2-en-2-yl,
cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyls such as but-1-en-1-yl,
but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl,
buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl,
cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.; and the like.

[0035]"Alkynyl" by itself or as part of another substituent refers to an
unsaturated branched, straight-chain or cyclic alkyl having at least one
carbon-carbon triple bond derived by the removal of one hydrogen atom
from a single carbon atom of a parent alkyne. Typical alkynyl groups
include, but are not limited to, ethynyl; propynyls such as
prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl,
but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.

[0036]"Alkyldiyl" by itself or as part of another substituent refers to a
saturated or unsaturated, branched, straight-chain or cyclic divalent
hydrocarbon group having the stated number of carbon atoms (i.e., C1-C6
means from one to six carbon atoms) derived by the removal of one
hydrogen atom from each of two different carbon atoms of a parent alkane,
alkene or alkyne, or by the removal of two hydrogen atoms from a single
carbon atom of a parent alkane, alkene or alkyne. The two monovalent
radical centers or each valency of the divalent radical center can form
bonds with the same or different atoms. Typical alkyldiyl groups include,
but are not limited to, methandiyl; ethyldiyls such as ethan-1,1-diyl,
ethan-1,2-diyl, ethen-1,1-diyl, ethen-1,2-diyl; propyldiyls such as
propan-1,1-diyl, propan-1,2-diyl, propan-2,2-diyl, propan-1,3-diyl,
cyclopropan-1,1-diyl, cyclopropan-1,2-diyl, prop-1-en-1,1-diyl,
prop-1-en-1,2-diyl, prop-2-en-1,2-diyl, prop-1-en-1,3-diyl,
cycloprop-1-en-1,2-diyl, cycloprop-2-en-1,2-diyl,
cycloprop-2-en-1,1-diyl, prop-1-yn-1,3-diyl, etc.; butyldiyls such as,
butan-1,1-diyl, butan-1,2-diyl, butan-1,3-diyl, butan-1,4-diyl,
butan-2,2-diyl, 2-methyl-propan-1,1-diyl, 2-methyl-propan-1,2-diyl,
cyclobutan-1,1-diyl; cyclobutan-1,2-diyl, cyclobutan-1,3-diyl,
but-1-en-1,1-diyl, but-1-en-1,2-diyl, but-1-en-1,3-diyl,
but-1-en-1,4-diyl, 2-methyl-prop-1-en-1,1-diyl,
2-methanylidene-propan-1,1-diyl, buta-1,3-dien-1,1-diyl,
buta-1,3-dien-1,2-diyl, buta-1,3-dien-1,3-diyl, buta-1,3-dien-1,4-diyl,
cyclobut-1-en-1,2-diyl, cyclobut-1-en-1,3-diyl, cyclobut-2-en-1,2-diyl,
cyclobuta-1,3-dien-1,2-diyl, cyclobuta-1,3-dien-1,3-diyl,
but-1-yn-1,3-diyl, but-1-yn-1,4-diyl, buta-1,3-diyn-1,4-diyl, etc.; and
the like. Where specific levels of saturation are intended, the
nomenclature alkanyldiyl, alkenyldiyl and/or alkynyldiyl is used. Where
it is specifically intended that the two valencies be on the same carbon
atom, the nomenclature "alkylidene" is used. A "lower alkyldiyl" is an
alkyldiyl group containing 1 to 6 carbon atoms. In some embodiments the
alkyldiyl groups are saturated acyclic alkanyldiyl groups in which the
radical centers are at the terminal carbons, e.g., methandiyl (methano);
ethan-1,2-diyl (ethano); propan-1,3-diyl (propano); butan-1,4-diyl
(butano); and the like (also referred to as alkylenes, defined infra).

[0037]"Alkylene" by itself or as part of another substituent refers to a
straight-chain saturated or unsaturated alkyldiyl group having two
terminal monovalent radical centers derived by the removal of one
hydrogen atom from each of the two terminal carbon atoms of
straight-chain parent alkane, alkene or alkyne. The locant of a double
bond or triple bond, if present, in a particular alkylene is indicated in
square brackets. Typical alkylene groups include, but are not limited to,
methylene (methano); ethylenes such as ethano, etheno, ethyno; propylenes
such as propano, prop[1]eno, propa[1,2]dieno, prop[1]yno, etc.; butylenes
such as butano, but[1]eno, but[2]eno, buta[1,3]dieno, but[1]yno,
but[2]yno, buta[1,3]diyno, etc.; and the like. Where specific levels of
saturation are intended, the nomenclature alkano, alkeno and/or alkyno is
used. In some embodiments, the alkylene group is (C1-C6) or (C1-C3)
alkylene. In some embodiments, the alkylene group is a straight-chain
saturated alkano group, e.g., methano, ethano, propano, butano, and the
like.

[0038]"Cycloalkyl" by itself or as part of another substituent refers to a
cyclic version of an "alkyl" group. Typical cycloalkyl groups include,
but are not limited to, cyclopropyl; cyclobutyls such as cyclobutanyl and
cyclobutenyl; cyclopentyls such as cyclopentanyl and cyclopentenyl;
cyclohexyls such as cyclohexanyl and cyclohexenyl; and the like.

[0040]"Aryl" by itself or as part of another substituent refers to a
monovalent aromatic hydrocarbon group having the stated number of carbon
atoms (i.e., C5-C15 means from 5 to 15 carbon atoms) derived by the
removal of one hydrogen atom from a single carbon atom of a parent
aromatic ring system. Typical aryl groups include, but are not limited
to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene,
anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene,
hexacene, hexaphene, hexylene, as-indacene, s-indacene, indane, indene,
naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene,
pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene,
picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene,
trinaphthalene, and the like, as well as the various hydro isomers
thereof. In some embodiments, the aryl group is (C5-C15) aryl, with
(C5-C10) being more typical. Specific examples are phenyl and naphthyl.

[0041]"Halogen" or "Halo" by themselves or as part of another substituent,
unless otherwise stated, refer to fluoro, chloro, bromo and iodo.

[0042]"Haloalkyl" by itself or as part of another substituent refers to an
alkyl group in which one or more of the hydrogen atoms are replaced with
a halogen. Thus, the term "haloalkyl" is meant to include monohaloalkyls,
dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls. For example, the
expression "(C1-C2) haloalkyl" includes fluoromethyl, difluoromethyl,
trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl, 1,2-difluoroethyl,
1,1,1-trifluoroethyl, perfluoroethyl, etc.

[0043]"Hydroxyalkyl" by itself or as part of another substituent refers to
an alkyl group in which one or more of the hydrogen atoms are replaced
with a hydroxyl substituent. Thus, the term "hydroxyalkyl" is meant to
include monohydroxyalkyls, dihydroxyalkyls, trihydroxyalkyls, etc.

[0044]The above-defined groups may include prefixes and/or suffixes that
are commonly used in the art to create additional well-recognized
substituent groups. As examples, "alkyloxy" or "alkoxy" refers to a group
of the formula --OR, "alkylamine" refers to a group of the formula --NHR
and "dialkylamine" refers to a group of the formula --NRR, where each R
is independently an alkyl. As another example, "haloalkoxy" or
"haloalkyloxy" refers to a group of the formula --OR', where R' is a
haloalkyl.

[0045]"Prodrug" refers to a derivative of an active compound (drug) that
may require a transformation under the conditions of use, such as within
the body, to release the active drug. Prodrugs are frequently, but not
necessarily, pharmacologically inactive until converted into the active
drug. Prodrugs are typically obtained by masking a functional group in
the drug compound believed to be in part required for activity with a
progroup (defined below) to form a promoiety which undergoes a
transformation, such as cleavage, under the specified conditions of use
to release the functional group, and hence the active drug. The cleavage
of the promoiety may proceed spontaneously, such as by way of a
hydrolysis reaction, or it may be catalyzed or induced by another agent,
such as by an enzyme, by light, by acid or base, or by a change of or
exposure to a physical or environmental parameter, such as a change of
temperature. The agent may be endogenous to the conditions of use, such
as an enzyme present in the cells to which the prodrug is administered or
the acidic conditions of the stomach, or it may be supplied exogenously.

[0046]A wide variety of progroups, as well as the resultant promoieties,
suitable for masking functional groups in the active stereoisomerically
enriched compounds described herein to yield prodrugs are well-known in
the art. For example, a hydroxyl functional group may be masked as a
sulfonate, ester or carbonate promoiety, which may be hydrolyzed in vivo
to provide the hydroxyl group. An amino functional group may be masked as
an amide, carbamate, imine, urea, phosphenyl, phosphoryl or sulfenyl
promoiety, which may be hydrolyzed in vivo to provide the amino group. A
carboxyl group may be masked as an ester (including silyl esters and
thioesters), amide or hydrazide promoiety, which may be hydrolyzed in
vivo to provide the carboxyl group. Other specific examples of suitable
progroups and their respective promoieties will be apparent to those of
skill in the art.

[0047]"Progroup" refers to a type of protecting group that, when used to
mask a functional group within an active stereoisomerically enriched drug
compound to form a promoiety, converts the drug into a prodrug. Progroups
are typically attached to the functional group of the drug via bonds that
are cleavable under specified conditions of use. Thus, a progroup is that
portion of a promoiety that cleaves to release the functional group under
the specified conditions of use. As a specific example, an amide
promoiety of the formula --NH--C(O)CH3 comprises the progroup
--C(O)CH3.

[0048]"Proliferative disorder" refers to a disease or disorder
characterized by aberrant cell proliferation, for example, where cells
divide more than their counterpart normal cells. The aberrant
proliferation may be caused by any mechanism of action or combination of
mechanisms of action. For example, the cell cycle of one or more cells
may be affected such that cell(s) divide more frequently than their
counterpart normal cells, or as another example, one or more cells may
bypass inhibitory signals, which would normally limit their number of
divisions. Proliferative diseases include, but are not limited to, slow
or fast growing tumors and cancers.

[0049]"Antiproliferative compound" refers to a compound that inhibits the
proliferation of a cell as compared to an untreated control cell of a
similar type. The inhibition can be brought about by any mechanism or
combination of mechanisms, and may operate to inhibit proliferation
cytostatically or cytotoxically. As a specific example, inhibition as
used herein includes, but is not limited to, arrest of cell division, a
reduction in the rate of cell division, proliferation and/or growth
and/or induction of cell death, by any mechanism of action, including,
for example apoptosis.

[0050]"Aurora kinase" refers to a member of the family of serine/threonine
protein kinases that are generally referred to as "Aurora" kinases. The
Aurora family of serine/threonine protein kinases are essential for cell
proliferation (see, e.g., Bischhoff & Plowman, 1999, Trends Cell Biol.
9:454-459; Giet & Prigent, 1999, J. Cell Science 112:3591-3601; Nigg,
2001, Nat. Rev. Mol. Cell. Biol. 2:21-32; Adams et al., 2001, Trends Cell
Biol. 11:49-54). Presently, there are three known mammalian family
members: Aurora-A ("2"), Aurora-B ("1") and Aurora-C ("3") (see, e.g.,
Giet & Prigent, 1999, J. Cell Sci. 112:3591-3601; Bischoff & Plowman,
1999, Trends Cell Biol. 9:454-459). As used herein, "Aurora kinase"
includes not only these three known mammalian family members, but also
later-discovered mammalian family members and homologous proteins from
other species and organisms (for non-limiting examples of homologous
members of the Aurora kinase family from other species and organisms see
Schumacher et al., 1998, J. Cell Biol. 143:1635-1646; Kimura et al.,
1997, J. Biol. Chem. 272:13766-13771).

[0051]"Aurora kinase-mediated process" or "Aurora kinase-mediated disease
or disorder" refers to a cellular process, disease or disorder in which
an Aurora kinase plays a role. The Aurora kinases are believed to play a
key role in protein phosphorylation events that regulate the mitotic
phase of the cell cycle. The human Aurora kinases display distinct
subcellular locations during mitosis. For example, Aurora-A is
upregulated during the M phase of the cell cycle and localizes to the
spindle pole during mitosis, suggesting involvement in centrosomal
functions. While Aurora-A activity is maximized during prophase, Aurora-B
is believed to play an important role during chromatid separation and
formation of the cleavage furrow in anaphase and telophase. The role of
Aurora-C is less clear, but it has been shown to localize to centrosomes
during mitosis from anaphase to cytokinesis. Moreover, inhibition of
Aurora kinase activity in mammalian cells leads to abnormal cell growth
and polyploidy (Terada et al., 1998, EMBO J. 17:667-676). Thus, Aurora
kinases are thought to regulate cell division, chromosome segregation,
mitotic spindle formation, and cytokinesis. As used herein, all of these
various processes are within the scope of "Aurora kinases-mediated
process."

[0052]Moreover, since its discovery in 1997, the mammalian Aurora kinase
family has been closely linked to tumorigenesis. The most compelling
evidence for this is that over-expression of Aurora-A transforms rodent
fibroblasts (Bischoff et al., 1998, EMBO J. 17:3052-3065). Cells with
elevated levels of this kinase contain multiple centrosomes and
multipolar spindles, and rapidly become aneuploid. The oncogenic activity
of Aurora kinases is likely to be linked to the generation of such
genetic instability. Indeed, a correlation between amplification of the
aurora-A locus and chromosomal instability in mammary and gastric tumors
has been observed (Miyoshi et al., 2001, Int. J. Cancer 92:370-373;
Sakakura et al., 2001, Brit. J. Cancer 84:824-831).

[0054]In contrast, the Aurora family is expressed at a low level in the
majority of normal tissues, the exceptions being tissues with a high
proportion of dividing cells, such as the thymus and testis (Bischoff et
al., 1998, EMBO J., 17:3052-3065).

[0056]Although over-expression of proteins by cancer cells is not always
indicative that inhibition of the protein activity will yield anti-tumor
effect, it has been confirmed in functional assays that at least the
following types of tumor cells are sensitive to inhibition of Aurora
kinase activity: prostate (DU145), cervical (Hela), pancreatic
(Mia-Paca2, BX-PC3), histological leukemia (U937), lung adenocarinoma,
lung epidermoid, small lung cell carcinoma, breast, renal carcinoma,
MolT3 (all) and Molt4 (all).

[0057]Based on the established role of Aurora kinases in a variety of
cancers, examples of "Aurora kinases-mediated diseases and disorders"
include, but are not limited to, melanoma, leukemia, and solid tumor
cancers, such as, for example, colon, breast, gastric, ovarian, cervical,
melanoma, renal, prostate, lymphoma, neuroblastoma, pancreatic and
bladder cancers.

[0058]"Therapeutically effective amount" refers to an amount of a compound
sufficient to treat a specified disorder, or disease or one or more of
its symptoms. In reference to tumorigenic proliferative disorders, a
therapeutically effective amount comprises an amount sufficient to, among
other things, cause the tumor to shrink, or to decrease the growth rate
of the tumor.

[0059]In many situations, standard treatments for tumorigenic
proliferative disorder involves surgical interaction to remove the
tumor(s), either alone or in combination with drug (chemo) and/or
radiation therapies. As used herein, a "therapeutically effect amount" of
a compound is intended to include an amount of compound that either
prevents the recurrance of tumors in subjects that have had tumor(s)
surgically removed, or slows the rate of recurrance of tumor(s) in such
subjects.

[0060]Accordingly, as used herein, amounts of compounds that provide
therapeutic benefit adjunctive to another type of therapy, such as
surgical intervention and/or treatment with other antiproliferative
agents, including, for example, 5-fluorouracil, vinorelbine, taxol,
vinblastine, cisplatin, topotecan, etc.), are included within the meaning
of "therapeutically effective amount."

[0061]"Prophylactically effective amount" refers to an amount of a
compound sufficient to prevent a subject from developing a specified
disorder or disease. Typically, subjects in which prophylaxis is
practiced are not suffering from the specified disorder or disease, but
are recognized as being at an elevated risk for developing this disease
or disorder based factors such as, but not limited to, diagnostic markers
and family history.

[0063]Skilled artisans will appreciate that in structural formula (I), the
stereochemistry at carbons 1, 2, 3 and 4 is unspecified, such that the
compounds according to structural formula (I) include eight
diastereomers, illustrated by structural formulae (Ia)-(Ih), below:

##STR00007## ##STR00008##

[0064]The compounds of structural formula (I) also include two cis
racemates, represented by structural formulae (IIa) and (IIb), and two
trans racemates, represented by structural formulae (IIIa) and (IIIb),
below:

##STR00009##

[0065]The cis racemate of structural formula (IIa) can be referred to as
the 2-exo-3-exo racemate, and includes the (1R,2R,3S,4S) and
(1S,2S,3R,4R) diastereomers of structural formulae (Ia) and (Ib),
respectively. The cis racemate of structural formula (Ib) can be referred
to as the 2-endo-3-endo racemate, and includes the (1R,2S,3R,4S) and
(1S,2R,3S,4R) diastereomers of structural formulae (Ic) and (id),
respectively. As described in more detail in the Examples section, for
compounds in which R5 is fluoro, R1 is hydrogen, R2 is
4-methylpiperazin-1-yl and R3 is methyl, these two cis racemates
exhibit antiproliferative activity against a variety of different tumor
cell lines in in vitro antiproliferation assays. However, this
2-exo-3-exo racemate (racemate rl) is approximately twenty-fold more
potent than the corresponding 2-endo-3-endo racemate (racemate r2) in all
cell lines tested with both racemates. Moreover, it has been discovered
that the (1R,2R,3S,4S) diastereomer of racemate r1 is largely responsible
for the potency of the racemate r1. When tested as isolated
stercoisomers, this (1R,2R,3S,4S) diastereomer (designated the "a"
diastereomer) generally exhibited IC50's in the nanomolar range, whereas
the (1S,2S,3R,4R) diastereomer (designated the "b" enantiomer) generally
exhibited IC50's in the micromolar range against the same cell lines.
Thus, in general, the (1R,2R,3S,4S) diastereomer of this compound is
generally 1000-fold more potent than its corresponding (1S,2S,3R,4R)
enantiomer. It is also approximately 20-50 times more potent than the
corresponding 2-endo-3-endo r2 racemate in the cell lines tested. The
(1R,2R,3S,4S) diastereomer exhibited similarly superior results compared
to its (1S,2S,3R,4R) enantiomer in cell-based inhibition assays against
Aurora kinase B. Based on the observed potency of this (1R,2R,3S,4S)
diastereomer, it is expected that the full range of (1R,2R,3S,4S)
diastereomers according to structural formula (Ia) will exhibit similarly
superior potencies as compared to their corresponding (1S,2S,3R,4R)
enantiomers, 2-exo-3-exo racemates, 2-endo-3-endo racemates and other
corresponding diastereomers.

[0066]Accordingly, provided herein are compounds that are enriched in this
particularly potent (1R,2R,3S,4S) diastereomer. In one embodiment, such
stereoisomerically enriched compounds include compounds according to
structural formula (I):

##STR00010##

[0067]that are enriched in the corresponding diastereomer of structural
formula (Ia):

[0072]each R4 is independently selected from the group consisting of
hydrogen, lower alkyl, arylalkyl, --ORa, --NRcRc,
--C(O)Ra, --C(O)ORa and --C(O)NRcRc; [0073]R5 is
hydrogen, halo, fluoro, --CN, --NO2, --C(O)ORa, or --CF3;
[0074]each n is independently an integer from 1 to 3; [0075]each Ra
is independently selected from the group consisting of hydrogen, lower
alkyl and lower cycloalkyl; [0076]each Rb is independently selected
from the group consisting of --ORa, --CF3, --OCF3,
--NRcRc, --C(O)Ra, --C(O)ORa, --C(O)NRcRc
and C(O)NRaRd; [0077]each Rc is independently selected
from the group consisting of hydrogen and lower alkyl, or, alternatively,
two Rc substituents may be taken together with the nitrogen atom to
which they are bonded to form a 4-9 membered saturated ring which
optionally includes 1-2 additional heteroatomic groups selected from O,
NRa, NRa--C(O)Ra, NRa--C(O)ORa and
NRa--C(O)NRa; and [0078]each Rd is independently lower
mono-hydroxyalkyl or lower di-hydroxyalkyl.

[0079]In another embodiment, such stereoisomerically enriched compounds
include 2-exo-3-exo cis racemates according to structural formula (IIa),
wherein R1, R2, R3, R4 and R5 are as previously
defined for structural formula (I), that are enriched in the diastereomer
of structural formula (Ia), supra.

[0080]As used herein, a compound is "enriched" in a particular
diastereomer when that diastereomer is present in excess over any other
diastereomer present in the compound. The actual percentage of the
particular diastereomer comprising the compound will depend upon the
number of other diastereomers present. As a specific example, a racemic
mixture is "enriched" in a specified enantiomer when that enantiomer
constitutes greater than 50% of the mixture. Regardless of the number of
diastereomers present, a compound that is enriched in a particular
diastereomer will typically comprise at least about 60%, 70%, 80%, 90%,
or even more, of the specified diastereomer. The amount of enrichment of
a particular diastereomer can be confirmed using conventional analytical
methods routinely used by those of skill in the art, as will be discussed
in more detail, below.

[0081]In another embodiment, the stereoisomerically enriched compounds
include compounds according to structural formula (Ia), supra, wherein
R1, R2, R3, R4 and R5 are as previously defined
for structural formula (I), that are substantially free of the
corresponding enantiomer and/or any other corresponding diastereomer. By
"substantially free of" is meant that the compound comprises less than
about 10% of the undesired diastereomers and/or enantiomers as
established using conventional analytical methods routinely used by those
of skill in the art (discussed in more detail below). In some
embodiments, the amount of undesired stereoisomers may be less than 10%,
for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or even less.
Stereoisomerically enriched compounds that contain about 95% or more of
the desired stereoisomer are referred to herein as "substantially pure"
stereoisomers. Stereoisomerically enriched compounds that contain about
99% or more of the desired stereoisomer are referred to herein as "pure"
stereoisomers. The purity of any stereoisomerically enriched compound
(diastereoisomeric purity; % de) can be confirmed using conventional
analytical methods, as will be described in more detail, below.

[0082]In some embodiments of the various stereoisomerically enriched
compounds described herein, R1 is hydrogen; R2 is

##STR00014##

and R3 is other than

##STR00015##

In other embodiments of the various stereoisomerically enriched compounds
described herein, R3 is hydrogen, methyl, methoxy, trifluoromethyl
or chloro. In still other embodiments, R4 is methyl, --C(O)CH3,
--C(O)OCH3 or --C(O)OCH2CH3.

[0083]In still other embodiments of the various stereoisomerically
enriched compounds described herein, R1 is hydrogen, R2 is
other than

[0085]In still other embodiments of the various stereoisomerically
enriched compounds described herein, R2 is other than

##STR00018##

and R3 is other than

##STR00019##

In still other embodiments, R1 and R2 are each hydrogen and
R3 is --OCH2NHRa. In some other embodiments, R1,
R2 and R3 are each, independently of one another selected from
the group consisting of hydrogen, methyl, methoxy, trifluoromethyl and
chloro, with the proviso that at least two of R1, R2 and
R3 are other than hydrogen.

[0086]In still other embodiments, R1 is hydrogen, R2 is selected
from the group consisting of hydrogen,

##STR00020##

and R3 is selected from the group consisting of hydrogen, lower
alkyl, halo, --CF3,

##STR00021##

In still other embodiments, R3 is selected from the group consisting
of hydrogen, methyl, chloro, --CF3,

##STR00022##

and R4 is methyl, --CORa or --CO(O)Ra where Ra is
methyl or ethyl. In yet another embodiment, R2 is selected from the
group consisting of hydrogen,

##STR00023##

and R3 is selected from the group consisting of hydrogen, lower
alkyl, halo, --CF3,

##STR00024##

In still other embodiments, R3 is selected from the group consisting
of hydrogen, methyl, chloro, --CF3

##STR00025##

and R4 is methyl, --CORa or --CO(O)Ra wherein Ra is
methyl or ethyl. Preferably, R2 is

##STR00026##

R4 is --CORa wherein Ra is methyl; and R3 is hydrogen.
In other embodiments, R2 is

##STR00027##

R4 is --CO(O)Ra wherein Ra is ethyl, and R3 is
hydrogen. In still another embodiment, R2 is

##STR00028##

and R3 is hydrogen.

[0087]In yet another embodiment, R2 is hydrogen; R3 is

##STR00029##

and R4 is methyl, --CORa or CO(O)Ra where Ra is methyl
or ethyl. Preferably, R2 is

##STR00030##

R4 is methyl and R3 is selected from the group consisting of
hydrogen, methyl, chloro and --CF3. More preferably, R3 is
methyl.

[0088]In still other embodiments of the stereoisomerically enriched
compounds described herein, R5 is fluoro.

[0090]Additional exemplary embodiments of compounds according to
structural formula (I) that may be stereoisomerically enriched in the
corresponding diastereomer of structural formula (Ia), supra,
substantially free of any enantiomers and/or diastereomer thereof, and/or
substantially pure or pure in the diastereomer of structural formula
(Ia), supra, are illustrated in TABLE 1, below:

[0091]When specific diastereomers and/or racemic mixtures of specific
compounds described herein, such as the compounds described in TABLE1,
are intended, the compound number is followed by a letter specifying the
specific diastereomer or racemic mixture as follows:

a=(1R,2R,3S,4S)

b=(1S,2S,3R,4R)

c=(1R,2S,3R,4S)

d=(1S,2R,3S,4R)

e=(1R,2R,3R,4S)

f=(1S,2S,3S,4R)

g=(1R,2S,3S,4S)

h=(1S,2R,3R,4R)

r1=2-exo-3-exo cis racemate

r2=2-endo-3-endo cis racemate

r3=2-exo-3-endo trans racemate

r4=2-endo-3-exo trans racemate

[0092]Thus, as a specific example, the (1R,2R,3S,4S) diastereomer of
compound 60 is referred to as compound 60a.

[0093]Those of skill in the art will appreciate that the
stereoisomerically enriched compounds described herein may include
functional groups that can be masked with progroups to create prodrugs.
Such prodrugs are usually, but need not be, pharmacologically inactive
until converted into their active drug form. For example, ester groups
commonly undergo acid-catalyzed hydrolysis to yield the parent carboxylic
acid when exposed to the acidic conditions of the stomach, or
base-catalyzed hydrolysis when exposed to the basic conditions of the
intestine or blood. Thus, when administered to a subject orally,
stereoisomerically enriched compounds that include ester moieties may be
considered prodrugs of their corresponding carboxylic acid, regardless of
whether the ester form is pharmacologically active.

[0094]Included within the scope of the invention are prodrugs of the
various stereoisomerically enriched compounds described herein. In such
prodrugs, any available functional moiety may be masked with a progroup
to yield a prodrug. Functional groups within the stereochemically
enriched compounds described herein that may be masked with progroups for
inclusion in a promoiety include, but are not limited to, amines (primary
and secondary), hydroxyls, sulfanyls (thiols), carboxyls, etc. Myriad
progroups suitable for masking such functional groups to yield
promoieties that are cleavable under the desired conditions of use are
known in the art. All of these progroups, alone or in combinations, may
be included in the stereoisomerically enriched prodrugs of the invention.

[0095]In one illustrative embodiment, the stereoisomerically enriched
prodrugs are compounds according to structural formulae (I), supra, in
which Ra, Rb and Rc may be, in addition to their
previously-defined alternatives, a progroup, that are enriched in the
corresponding diastereomer of structural formula (Ia), supra.

[0096]Those of skill in the art will appreciate that many of the compounds
and prodrugs described herein, as well as the various compound species
specifically described and/or illustrated herein, may exhibit the
phenomena of tautomerism and conformational isomerism. For example, the
compounds and prodrugs may exist in several tautomeric forms, including
the enol form, the keto form and mixtures thereof. As the various
compound names, formulae and compound drawings within the specification
and claims can represent only one of the possible tautomeric or
conformational forms, it should be understood that the invention
encompasses any tautomers or conformational isomers, of the compounds or
prodrugs having one or more of the utilities described herein, as well as
mixtures of these various different isomeric forms. In cases of limited
rotation around the 2,4-pyrimidinediamine core structure, atrop isomers
are also possible and are also specifically included in the compounds
and/or prodrugs of the invention.

[0097]Depending upon the nature of the various substituents, the
stereoisomerically enriched compounds and prodrugs may be in the form of
salts. Such salts include salts suitable for pharmaceutical uses
("pharmaceutically-acceptable salts"), salts suitable for veterinary
uses, etc. Such salts may be derived from acids or bases, as is
well-known in the art.

[0099]Pharmaceutically acceptable salts also include salts formed when an
acidic proton present in the parent compound is either replaced by a
metal ion (e.g., an alkali metal ion, an alkaline earth metal ion or an
aluminum ion) or coordinates with an organic base (e.g., ethanolamine,
diethanolamine, triethanolamine, N-methylglucamine, morpholine,
piperidine, dimethylamine, diethylamine, etc.).

[0100]The stereoisomerically enriched compounds and prodrugs, as well as
the salts thereof, may also be in the form of hydrates, solvates and/or
N-oxides, as are well-known in the art.

[0101]Stereoisomeric enrichment and/or purity of compounds and prodrug
described herein may be established by conventional analytical methods
well known to those of skill in the art. For example, use of chiral NMR
shift reagents, gas chromatographic analysis using chiral columns, high
pressure liquid chromatographic analysis using chiral columns, formation
of diastereomeric derivatives through reaction with chiral reagents and
conventional analysis may be used to establish the stereoisomeric
enrichment and/or purity of a specific stereoisomer. Alternatively,
synthesis using starting materials of known stereoisomeric enrichment
and/or purity may be used to establish the stereoisomeric enrichment
and/or purity of the compounds described herein. Other analytical methods
for demonstrating stereoisomeric homogeneity are well within the ambit of
the skilled artisan.

6.3 Methods of Synthesis

[0102]The stereoisomerically enriched compounds and prodrugs may be
synthesized via a variety of different synthetic routes using
commercially available starting materials and/or starting materials
prepared by conventional synthetic methods. A variety of exemplary
synthetic routes that can be used to synthesize the stereoisomerically
enriched compounds and prodrugs are described in WO 03/063794 and US
2004/0029902, the disclosures of which are incorporated herein by
reference.

[0103]For purposes of illustration, an exemplary synthetic scheme that can
be used to synthesize the full range of compounds described herein is
illustrated in Scheme (I), below:

##STR00048##

[0104]In Scheme (I), R1, R2, R3 and R5 are as
previously defined for structural formula (I), supra, X is a halogen
(e.g., F, Cl, Br or I), and each G is, independently of the other,
selected from O and S. It should be noted that an "*" in aminocarboxamide
6 indicates that the particular stereocenter is not specified.
Accordingly, those of skill in the art will appreciate that Scheme (I)
may be used to prepare racemic diastereomeric mixtures,
diastereomerically enriched mixtures of compounds according to structural
formula (I), as well as stereoisomers of the compounds of structural
formula (I) that are substantially free of other specified diastereomers.

[0105]Referring to Scheme (I), uracil or thiouracil 2 is dihalogenated at
the 2- and 4-positions using the standard halogenating agent POX3
(or other halogenating agents) under standard conditions to yield
2,4-bis-halo pyrimidine 4. The halide at the C4 position is more reactive
towards nucleophiles than the halide at the C2 position in pyrimidine 4.
This differential reactivity can be exploited to synthesize the compounds
and prodrugs described herein by first reacting 2,4-bis-halopyrimidine 4
with one equivalent of 2-aminobicyclo[2.2.1]hept-5-ene-3-carboxamide 6,
yielding 8, followed by reaction with aniline 10 to yield compounds
according to structural formula (I). Those of skill in the art will
appreciate that the stereoisomeric configuration and optical purity of
aminocarboxamide 6 will, in most circumstances, determine the
stereoisomeric configuration and optical purity of the compounds of
structural formula (I).

[0106]In most situations, the C4 halide is more reactive towards
nucleophiles, as illustrated in the Scheme. However, as will be
recognized by skilled artisans, the identity of the R5 substituent
may alter this reactivity. For example, when R5 is trifluoromethyl,
a 50:50 mixture of 4N-substituted-4-pyrimidineamine 8 and the
corresponding 2N-substituted-2-pyrimidineamine is obtained. Regardless of
the identity of the R5 substituent, the regioselectivity of the
reaction can be controlled by adjusting the solvent and other synthetic
conditions (such as temperature), as is well-known in the art.

[0107]The reactions depicted in Scheme (I) may proceed more quickly when
the reaction mixtures are heated via microwave. When heating in this
fashion, the following conditions may be used: heat to 175° C. in
ethanol for 5-20 min. in a Smith Reactor (Personal Chemistry, Biotage AB,
Sweden) in a sealed tube (at 20 bar pressure).

[0109]Anilines 10 may be purchased from commercial sources or,
alternatively, may be synthesized utilizing standard techniques. For
example, suitable anilines may be synthesized from nitro precursors using
standard chemistry. Specific exemplary reactions are provided in the
Examples section. See also Vogel, 1989, Practical Organic Chemistry,
Addison Wesley Longman, Ltd. and John Wiley & Sons, Inc.

[0110]Skilled artisans will recognize that in some instances anilines 10
may include functional groups that require protection during synthesis.
The exact identity of any protecting group(s) used will depend upon the
identity of the functional group being protected, and will be apparent to
these of skill in the art. Guidance for selecting appropriate protecting
groups, as well as synthetic strategies for their attachment and removal,
may be found, for example, in Greene & Wuts, Protective Groups in Organic
Synthesis, 3d Edition, John Wiley & Sons, Inc., New York (1999) and the
references cited therein (hereinafter "Greene & Wuts").

[0111]Prodrugs as described herein may be prepared by routine modification
of the above-described methods.

[0112]As skilled artisans will appreciate, the desired (1R,2R,3S,4S)
diastereomer corresponding to structural formula (Ia), supra, can be
isolated by chiral separation or other standard techniques. Methods for
chirally resolving specific diastereomers are described in more detail in
the Examples section.

[0113]Stereoisomerically enriched compounds and/or substantially pure
and/or pure diastereomers can also be synthesized from
2-amino-3-carboxamide starting materials 6 having specified
stereochemistry, or with the aid of chiral auxiliaries.

[0114]In one exemplary embodiment, illustrated in Scheme (II), below, the
desired diastereomer is resolved chemically using
(R)-methyl-p-methoxybenzylamine 18 as a chiral auxiliary.

##STR00049##

[0115]In Scheme (II), 2-exo-3-exo racemic β-lactam 14r1 (prepared as
described in Stajar et al., 1984, Tetrahedron 40(12): 2385) is protected
with a Boc group, yielding the corresponding racemic Boc-protected
β-lactam 16r1. Boc-protected racemate 16r1 is then reacted with
(R)-methyl-para-methoxybenzylamine 18, yielding a mixture of
diastereomers 20a and 20b. This diastereomeric mixture is treated with an
acid such as TFA to cleave the Boc group, yielding a mixture of
diastereomers 22a and 22b, which can be reacted with 2,4-dihalopyrimidine
4 to afford a racemic mixture of compounds 24a and 24b. At this stage,
compounds 24a and 24b can be separated from one another by
crystallization and reacted with aniline 10 to afford isolated
diastereomers 25a and 25b. The chiral auxiliaries from isolated
diasteromers 25a and 25b can then be cleaved to yield isolated
diastereomers according to structural formulae (Ia) and (Ib),
respectively.

[0116]For compounds 25a and 25b in which R1 is hydrogen, R2 is
4-methyl-piperazin-1-yl, R3 is methyl and R5 is fluoro,
cleavage of the chiral auxiliary proved difficult. For these and other
compounds where such cleavage proves difficult, the chiral auxiliary can
be cleaved from compounds 24a and 24b, and the resultant isolated
compounds reacted with aniline 10 to yield isolated diastereomers
according to structural formulae (Ia) and (Ib). Specific examples of such
reactions are described in the Examples section.

[0118]Examples of synthesizing specified diasteromers according to
structural formula (Ia) utilizing enzyme reactions are illustrated in
Schemes (III) and (IV), below. A specific example of the use of Novozyme
435 enzyme as illustrated in Scheme (IV), which like the Chirazyme enyme
discussed supra and illustrated in Scheme (III), can be used to resolve
enantiomers from racemic β-lactams, is described in the Examples
section.

##STR00050##

##STR00051##

6.4 Activity of the Antiproliferative Compounds

[0119]Active stereoisomerically enriched compounds typically inhibit
proliferation of desired cells, such as tumor cells, with an IC50 in
the range of about 20 μM or less, as measured in a standard in vitro
cellular proliferation assay. Of course, skilled artisans will appreciate
that compounds which exhibit lower IC50s, for example on the order
of 10 μM, 1 μM, 100 nM, 10 nM, 1 nM, or even lower, may be
particularly useful in therapeutic applications. The antiproliferative
activity may be cytostatic or it may be cytotoxic. In instances where
antiproliferative activity specific to a particular cell type is desired,
the compound may be assayed for activity with the desired cell type and
counter-screened for a lack of activity against other cell types. The
desired degree of "inactivity" in such counter screens, or the desired
ratio of activity vs. inactivity may vary for different situations, and
may be selected by the user.

[0120]Active compounds also typically inhibit an activity of an Aurora
kinase, with an IC50 in the range of about 20 μM or less,
typically in the range of about 10 μM, 1 μM, 100 nM, 10 mM, 1 mM,
or even lower. The IC50 against an aurora kinase can be determined
in a standard in vitro assay with an isolated aurora kinase, or in a
functional cellular array. A suitable enzyme coupled assay that can be
used to determine the degree of Aurora kinase activity is described in
Fox et al., 1998, Protein Sci. 7:2249-2255. Kemptide peptide sequence
LRRASLG (Bochern Ltd., UK) can be used as a substrate for Aurora kinase-A
Aurora kinase-B and/or Aurora kinase-C, and reactions can be carried out
at 30° C. in a solution containing 100 mM HEPES (pH 7.5), 10 mM Mg
Cl2, 25 mM NaCl, 1 mM DTT. IC50 values can be determined using
computerized non-linear regression with commercially-available software
(e.g., Prism 3.0, GraphPed Software, San Diego, Calif.). A suitable
cell-based functional assay is described in the Examples section.

6.5 Uses of the Antiproliferative Compounds

[0121]The active stereoisomerically enriched compounds, including the
various prodrugs, salts, hydrates and/or N-oxide forms thereof, may be
used to inhibit Aurora kinases, Aurora kinase-mediated processes, and/or
cell proliferation in a variety of contexts. According to some
embodiments, a cell or population of cells is contacted with an amount of
such a compound effective to inhibit an activity of an Aurora kinase, an
Aurora kinase-mediated process and/or proliferation of the cell or cell
population. When used to inhibit cellular proliferation, the compound may
act cytotoxically to kill the cell, or cytostatically to inhibit
proliferation without killing the cell.

[0122]In some embodiments, the methods may be practiced in vivo as a
therapeutic approach towards the treatment or prevention of Aurora
kinase-mediated diseases or disorders, and in particular proliferative
disorders. Thus, in a specific embodiment, the stereoisomerically
enriched compounds described herein, (and the various forms described
herein) may be used to treat or prevent proliferative disorders in animal
subjects, including humans. The method generally comprises administering
to the subject an amount of a stereoisomerically enriched compound, or a
prodrug, salt, hydrate or N-oxide thereof, effective to treat or prevent
the disorder. In one embodiment, the subject is a mammal, including, but
not limited to, bovine, horse, feline, canine, rodent, or primate. In
another embodiment, the subject is a human.

[0123]A variety of cellular proliferative disorders may be treated or
prevented with the compounds described herein. In some embodiments, the
compounds are used to treat various cancers in afflicted subjects.
Cancers are traditionally classified based on the tissue and cell type
from which the cancer cells originate. Carcinomas are considered cancers
arising from epithelial cells while sarcomas are considered cancers
arising from connective tissues or muscle. Other cancer types include
leukemias, which arise from hematopoietic cells, and cancers of nervous
system cells, which arise from neural tissue. For non-invasive tumors,
adenomas are considered benign epithelial tumors with glandular
organization while chondomas are benign tumor arising from cartilage. In
the present invention, the described compounds may be used to treat
proliferative disorders encompassed by carcinomas, sarcomas, leukemias,
neural cell tumors, and non-invasive tumors.

[0126]It is to be understood that the descriptions of proliferative
disorders is not limited to the conditions described above, but
encompasses other disorders characterized by uncontrolled growth and
malignancy. It is further understood that proliferative disorders include
various metastatic forms of the tumor and cancer types described herein.
The compounds of the present invention may be tested for effectiveness
against the disorders described herein, and a therapeutically effective
regimen established. Effectiveness, as further described below, includes
reduction or remission of the tumor, decreases in the rate of cell
proliferation, or cytostatic or cytotoxic effect on cell growth.

6.6 Combination Therapies

[0127]The stereoisomerically enriched compounds described herein may be
used alone, in combination with one another, or as an adjunct to, or in
conjunction with, other established antiproliferative therapies. Thus,
the compounds may be used with traditional cancer therapies, such as
ionization radiation in the form of γ-rays and x-rays, delivered
externally or internally by implantation of radioactive compounds, and as
a follow-up to surgical removal of tumors.

[0128]In another aspect, the compounds may be used with other
chemotherapeutic agents useful for the disorder or condition being
treated. These compounds may be administered simultaneously,
sequentially, by the same route of administration, or by a different
route.

[0129]In some embodiments, the present compounds are used with other
anti-cancer or cytotoxic agents. Various classes of anti-cancer and
anti-neoplastic compounds include, but are not limited to, alkylating
agents, antimetabolites, vinca alkyloids, taxanes, antibiotics, enzymes,
cytokines, platinum coordination complexes, substituted ureas, tyrosine
kinase inhibitors, hormones and hormone antagonists. Exemplary alkylating
agents include, by way of example and not limitation, mechlorothamine,
cyclophosphamide, ifosfamide, melphalan, chlorambucil, ethyleneimines,
methylmelamines, alkyl sulfonates (e.g., busulfan), and carmustine.
Exemplary antimetabolites include, by way of example and not limitation,
folic acid analog methotrexate; pyrimidine analog fluorouracil, cytosine
arbinoside; purine analogs mercaptopurine, thioguanine, and azathioprine.
Exemplary vinca alkyloids include, by way of example and not limitation,
vinblastine, vincristine, paclitaxel, and colchicine. Exemplary
antibiotics include, by way of example and not limitation, actinomycin D,
daunorubicin, and bleomycin. An exemplary enzyme effective as
anti-neoplastic agents include L-asparaginase. Exemplary coordination
compounds include, by way of example and not limitation, cisplatin and
carboplatin. Exemplary hormones and hormone related compounds include, by
way of example and not limitation, adrenocorticosteroids prednisone and
dexamethasone; aromatase inhibitors amino glutethimide, formestane, and
anastrozole; progestin compounds hydroxyprogesteron caproate,
medroxyprogesterone; and anti-estrogen compound tamoxifen.

[0131]Additional anti-proliferative compounds useful in combination with
the stereoisomerically enriched compounds described herein include, by
way of example and not limitation, antibodies directed against growth
factor receptors (e.g., anti-Her2); antibodies for activating T cells
(e.g., anti-CTLA-4 antibodies); and cytokines such as interferon-α
and interferon-γ, interleukin-2 and GM-CSF.

6.7 Formulations and Administration

[0132]When used to treat or prevent such diseases, the active compounds
and prodrugs may be administered singly, as mixtures of one or more
active compounds, or in mixture or combination with other agents useful
for treating such diseases and/or the symptoms associated with such
diseases. The active compounds and prodrugs may also be administered in
mixture or in combination with agents useful to treat other disorders or
maladies, such as steroids, membrane stabilizers. The active compounds or
prodrugs may be administered per se, or as pharmaceutical compositions
comprising an active compound or prodrug.

[0133]Pharmaceutical compositions comprising the active compounds (or
prodrugs thereof) may be manufactured by means of conventional mixing,
dissolving, granulating, dragee-making levigating, emulsifying,
encapsulating, entrapping or lyophilization processes. The compositions
may be formulated in conventional manner using one or more
physiologically acceptable carriers, diluents, excipients or auxiliaries
which facilitate processing of the active compounds into preparations
which can be used pharmaceutically (see Remington's Pharmaceutical
Sciences, 15th Ed., Hoover, J. E. ed., Mack Publishing Co. (2003)

[0134]The active compound or prodrug may be formulated in the
pharmaceutical compositions per se, or in the form of a hydrate, solvate,
N-oxide or pharmaceutically acceptable salt, as previously described.
Typically, such salts are more soluble in aqueous solutions than the
corresponding free acids and bases, but salts having lower solubility
than the corresponding free acids and bases may also be formed.

[0135]Pharmaceutical compositions may take a form suitable for virtually
any mode of administration, including, for example, topical, ocular,
oral, buccal, systemic, nasal, injection, transdermal, rectal, vaginal,
etc., or a form suitable for administration by inhalation or
insufflation.

[0136]For topical administration, the active compound(s) or prodrug(s) may
be formulated as solutions, gels, ointments, creams, suspensions, etc. as
are well-known in the art.

[0137]Systemic formulations include those designed for administration by
injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or
intraperitoneal injection, as well as those designed for transdermal,
transmucosal oral or pulmonary administration.

[0138]Useful injectable preparations include sterile suspensions,
solutions or emulsions of the active compound(s) in aqueous or oily
vehicles. The compositions may also contain formulating agents, such as
suspending, stabilizing and/or dispersing agent. The formulations for
injection may be presented in unit dosage form, e.g., in ampoules or in
multidose containers, and may contain added preservatives.

[0139]Alternatively, the injectable formulation may be provided in powder
form for reconstitution with a suitable vehicle, including but not
limited to sterile pyrogen free water, buffer, dextrose solution, etc.,
before use. To this end, the active compound(s) may be dried by any
art-known technique, such as lyophilization, and reconstituted prior to
use.

[0140]For transmucosal administration, penetrants appropriate to the
barrier to be permeated are used in the formulation. Such penetrants are
known in the art.

[0142]Liquid preparations for oral administration may take the form of,
for example, elixirs, solutions, syrups or suspensions, or they may be
presented as a dry product for constitution with water or other suitable
vehicle before use. Such liquid preparations may be prepared by
conventional means with pharmaceutically acceptable additives such as
suspending agents (e.g., sorbitol syrup, cellulose derivatives or
hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia);
non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol,
cremophoretm or fractionated vegetable oils); and preservatives (e.g.,
methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, preservatives, flavoring, coloring and
sweetening agents as appropriate.

[0143]Preparations for oral administration may be suitably formulated to
give controlled release of the active compound or prodrug, as is well
known in the art.

[0144]For buccal administration, the compositions may take the form of
tablets or lozenges formulated in conventional manner.

[0145]For rectal and vaginal routes of administration, the active
compound(s) may be formulated as solutions (for retention enemas)
suppositories or ointments containing conventional suppository bases such
as cocoa butter or other glycerides.

[0146]For nasal administration or administration by inhalation or
insufflation, the active compound(s) or prodrug(s) can be conveniently
delivered in the form of an aerosol spray from pressurized packs or a
nebulizer with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, fluorocarbons, carbon dioxide or other
suitable gas. In the case of a pressurized aerosol, the dosage unit may
be determined by providing a valve to deliver a metered amount. Capsules
and cartridges for use in an inhaler or insufflator (for example capsules
and cartridges comprised of gelatin) may be formulated containing a
powder mix of the compound and a suitable powder base such as lactose or
starch.

[0147]For ocular administration, the active compound(s) or prodrug(s) may
be formulated as a solution, emulsion, suspension, etc. suitable for
administration to the eye. A variety of vehicles suitable for
administering compounds to the eye are known in the art. Specific
non-limiting examples are described in U.S. Pat. No. 6,261,547; U.S. Pat.
No. 6,197,934; U.S. Pat. No. 6,056,950; U.S. Pat. No. 5,800,807; U.S.
Pat. No. 5,776,445; U.S. Pat. No. 5,698,219; U.S. Pat. No. 5,521,222;
U.S. Pat. No. 5,403,841; U.S. Pat. No. 5,077,033; U.S. Pat. No.
4,882,150; and U.S. Pat. No. 4,738,851.

[0148]For prolonged delivery, the active compound(s) or prodrug(s) can be
formulated as a depot preparation for administration by implantation or
intramuscular injection. The active ingredient may be formulated with
suitable polymeric or hydrophobic materials (e.g., as an emulsion in an
acceptable oil) or ion exchange resins, or as sparingly soluble
derivatives, e.g., as a sparingly soluble salt. Alternatively,
transdermal delivery systems manufactured as an adhesive disc or patch
which slowly releases the active compound(s) for percutaneous absorption
may be used. To this end, permeation enhancers may be used to facilitate
transdermal penetration of the active compound(s). Suitable transdermal
patches are described in for example, U.S. Pat. No. 5,407,713; U.S. Pat.
No. 5,352,456; U.S. Pat. No. 5,332,213; U.S. Pat. No. 5,336,168; U.S.
Pat. No. 5,290,561; U.S. Pat. No. 5,254,346; U.S. Pat. No. 5,164,189;
U.S. Pat. No. 5,163,899; U.S. Pat. No. 5,088,977; U.S. Pat. No.
5,087,240; U.S. Pat. No. 5,008,110; and U.S. Pat. No. 4,921,475.

[0149]Alternatively, other pharmaceutical delivery systems may be
employed. Liposomes and emulsions are well-known examples of delivery
vehicles that may be used to deliver active compound(s) or prodrug(s).
Certain organic solvents such as dimethylsulfoxide (DMSO) may also be
employed, although usually at the cost of greater toxicity.

[0150]The pharmaceutical compositions may, if desired, be presented in a
pack or dispenser device which may contain one or more unit dosage forms
containing the active compound(s). The pack may, for example, comprise
metal or plastic foil, such as a blister pack. The pack or dispenser
device may be accompanied by instructions for administration.

6.8 Effective Dosages

[0151]The active compound(s) or prodrug(s), or compositions thereof, will
generally be used in an amount effective to achieve the intended result,
for example in an amount effective to treat or prevent the particular
disease being treated. The compound(s) may be administered
therapeutically to achieve therapeutic benefit. By therapeutic benefit is
meant eradication or amelioration of the underlying disorder being
treated and/or eradication or amelioration of one or more of the symptoms
associated with the underlying disorder such that the patient reports an
improvement in feeling or condition, notwithstanding that the patient may
still be afflicted with the underlying disorder. Therapeutic benefit also
includes halting or slowing the progression of the disease, regardless of
whether improvement is realized.

[0152]The amount of compound administered will depend upon a variety of
factors, including, for example, the particular indication being treated,
the mode of administration, the severity of the indication being treated
and the age and weight of the patient, the bioavailability of the
particular active compound, etc. Determination of an effective dosage is
well within the capabilities of those skilled in the art.

[0153]Effective dosages may be estimated initially from in vitro assays.
For example, an initial dosage for use in animals may be formulated to
achieve a circulating blood or serum concentration of active compound
that is at or above an IC50 of the particular compound as measured
in an in vitro assay, such as the in vitro assays described in the
Examples section. Calculating dosages to achieve such circulating blood
or serum concentrations taking into account the bioavailability of the
particular compound is well within the capabilities of skilled artisans.
For guidance, the reader is referred to FingI & Woodbury, "General
Principles," In: Goodman and Gilman's The Pharmaceutical Basis of
Therapeutics, Chapter 1, pp. 1-46, latest edition, Pergamon Press, and
the references cited therein.

[0154]Initial dosages may also be estimated from in vivo data, such as
animal models. Animal models useful for testing the efficacy of compounds
to treat or prevent the various diseases described above are well-known
in the art. Dosage amounts will typically be in the range of from about
0.0001 or 0.001 or 0.01 mg/kg/day to about 100 mg/kg/day, but may be
higher or lower, depending upon, among other factors, the activity of the
compound, its bioavailability, the mode of administration and various
factors discussed above. Dosage amount and interval may be adjusted
individually to provide plasma levels of the compound(s) which are
sufficient to maintain therapeutic or prophylactic effect. For example,
the compounds may be administered once per week, several times per week
(e.g., every other day), once per day or multiple times per day,
depending upon, among other things, the mode of administration, the
specific indication being treated and the judgment of the prescribing
physician. In cases of local administration or selective uptake, such as
local topical administration, the effective local concentration of active
compound(s) may not be related to plasma concentration. Skilled artisans
will be able to optimize effective local dosages without undue
experimentation.

[0155]Preferably, the compound(s) will provide therapeutic or prophylactic
benefit without causing substantial toxicity. Toxicity of the compound(s)
may be determined using standard pharmaceutical procedures. The dose
ratio between toxic and therapeutic (or prophylactic) LD50/ED50
effect is the therapeutic index (LD50 is the dose lethal to 50% of
the population and ED50 is the dose therapeutically effective in 50%
of the population). Compounds(s) that exhibit high therapeutic indices
are preferred.

6.9 Kits

[0156]The compounds and/or prodrugs described herein may be assembled in
the form of kits. In some embodiments, the kit provides the compound(s)
and reagents to prepare a composition for administration. The composition
may be in a dry or lyophilized form, or in a solution, particularly a
sterile solution. When the composition is in a dry form, the reagent may
comprise a pharmaceutically acceptable diluent for preparing a liquid
formulation. The kit may contain a device for administration or for
dispensing the compositions, including, but not limited to syringe,
pipette, transdermal patch, or inhalant.

[0157]The kits may include other therapeutic compounds for use in
conjunction with the compounds described herein. In some embodiments, the
therapeutic agents are other anti-cancer and anti-neoplastic compounds.
These compounds may be provided in a separate form, or mixed with the
compounds of the present invention.

[0158]The kits will include appropriate instructions for preparation and
administration of the composition, side effects of the compositions, and
any other relevant information. The instructions may be in any suitable
format, including, but not limited to, printed matter, videotape,
computer readable disk, or optical disc.

7. EXAMPLES

[0159]The inventions are further defined by reference to the following
examples, which describe the preparation of the various compounds
described herein, methods for assaying their biological activity, and
methods for their use. It will be apparent to the skilled artisan that
many modifications, both to the materials and methods may be practiced
without departing from the scope of the inventions.

[0165]Procedure: Part 1: A solution of 2,5-norbornadiene 47 (25.0 mL,
0.246 mole) in CH2Cl2 (110 mL, fresh bottle) was cooled in an
ice/NaCl bath (-10° C.). To this was added drop-wise a solution of
CSI (21.4 mL, 0.246 mole) in CH2Cl2 (45 mL, fresh bottle) at a
rate to maintain the temperature below 5° C. (the addition took
approx. 1.25 hr.). Upon completion of the addition, the reaction mixture
was stirred for 1 hour at 0-5° C. and then removed from the
cooling bath and allowed to warm to 20° C. The reaction mixture
was quenched with water (60 mL) and vigorously stirred for several
minutes. The organic layer was separated, washed with brine, and dried
with Na2SO4. Concentration gave light brown oil.

[0166]Part 2: A mixture of Na2SO3 (24.5 g), water (70 mL), and
CH2Cl2 (30 mL) was cooled in an ice/NaCl bath. The oil from
Part 1 was diluted to 100 mL with CH2Cl2 and added dropwise to
the above mixture at a rate to maintain the temperature below 15°
C. (the addition took approx. 1.75 hr). The pH of the reaction mixture
was monitored with a pH meter and kept basic (pH 7-10) by adjusting with
10% NaOH (w/v) (as needed). Upon completion of the addition, the reaction
mixture was stirred for 1 hour at 5-10° C. (final pH was 8.5). The
reaction mixture was poured into a separatory funnel and the
CH2Cl2 layer separated. This organic phase was a thick and
gelatinous solid suspension. It was diluted with water (approx. 400 mL)
to make a more free flowing solution. The aqueous layer was further
extracted with CH2Cl2 (4×100 mL). (Alternatively, the
solids can be separated from the CH2Cl2 by centrifugation. The
solids can then be diluted with water (until almost all dissolved) and
extracted with CH2Cl2). The aqueous layer was further extracted
with CH2Cl2 (10×100 mL). The CH2Cl2 extracts
were monitored by TLC for the presence of product. The combined organic
extracts were washed with brine, dried with MgSO4, and filtered
through celite. Removal of solvent gave the desired product,
racemic-2-exo-3-endo 3-aza-4-oxo-tricyclo[4.2.1.0(2,5)]non-7-ene 14r1 as
white solid (20.5 g, 62%). 1H NMR (DMSO-d6): δ 8.01 (bs,
1H), 6.22 (dd, J=3.3 and 5.4 Hz, 1H), 6.12 (dd, J=3.3 and 5.4 Hz, 1H),
2.88 (dd, J=1.5 and 3.3, 1H), 2.79 (bs, 1H), 2.74 (bs, 1H), 1.58 (d,
J=9.3 Hz, 1H), and 1.47 (d, J=9.3 Hz, 1H).

[0169]A racemic mixture of the title compound was prepared from the
2-exo-3-exo racemate of 2-aminobicylco[2.2.1]hept-5-ene-3-carboxamide as
follows.

[0170]Reaction:

##STR00056##

[0171]Procedure: A round bottom flask equipped with a rubber septum and a
magnetic stirring bar was charged with racemic N--BOC-β-lactam 16r1
(2.0 g) under a positive pressure of nitrogen. To this were added ethyl
acetate (25 mL) followed by 30% ammonia in water (25 mL) and stirred at
room temperature for 3 hours. The ethyl acetate layer was separated and
washed with 5% aqueous solution of NaHCO3 (20 mL), dried over
anhydrous Na2SO4 and solvent was evaporated to afford 1.10 gm
of racemic N--BOC carboxyamide 28r1.

[0172]Reaction:

##STR00057##

[0173]Procedure: A round bottom flask equipped with N2 inlet and a
magnetic stirring bar was charged with racemic N--BOC lactam 28r1 (2.00
g, 7.9 mmol) and then treated with 20% of TFA in CH2Cl2 at room
temperature for 2 hours. The resulting solution was concentrated under a
reduced pressure. The trace of TFA was removed under high vacuum for
several hours to afford the intermediate, TFA salt (30r1, racemic). The
resulting racemic TFA salt 30r1 was treated with
2,4-dichloro-5-fluoropyrimidine 10 (1.58 g, 9.51 mm) in MeOH:H2O
(20:10 mL) in the presence of NaHCO3 (1.33 g, 15.84 mmol) at room
temperature for 48 hours. The reaction mixture was diluted with H2O
(25 mL), satured with NaCl and extracted with EtOAc (3×50 mL). Upon
drying over anhydrous Na2SO4, the solvent was evaporated and
the residue was chromatographed (silica gel, CH2Cl2 then 2-4%
2N NH3/MeOH in CH2Cl2) to afford 1.3 g of racemic
mono-SNAr product 36r1.

[0176]Isolation of Enantionmers: The diastereomers were resolved and
isolated from racemate 60r1 by chiral preparative HPLC chromatography
Phenomenex Chirex 3020 250×10 mm column), eluting with a 35:63:2
(vol:vol:vol) mixture of hexane:dichloromethane:methanol at a flow rate
of 6 mL/min. The enantiomer eluting at 9.44 min. was designated the E1
enantiomer and the enantiomer eluting at 12.74 min. was designated the E2
enantiomer.

[0187]Procedure: Immobilized Lipolase (8.0 g, from Sigma, order number
L4777), β-lactam 14r1 (racemic: 2-exo-3-exo) (4.0 g, 7.4 mmol) and
water (0.13 ml, 7.4 mmol) were added to 250 ml diisopropyl ether in a
pressure flask. The mixture was degassed with nitrogen for 20 minutes and
the flask was sealed and incubated for 14 days at 70° C. The
mixture was cooled to room temperature, filtered over celite and washed
with 300 ml diisopropyl ether. The combined filtrate was concentrated to
dryness and the residue was crystallized from diisopropyl ether to give
the enantiomerically pure β-lactam 14a as colorless needles (1.22 g,
61%). The enantiomeric purity was greater than 99% as determined by
chiral HPLC.

[0189]Procedure: A homogeneous mixture of enantiomerically pure
3-aza-4-oxo-tricyclo[4.2.1.0(2,5)]non-7-ene 14a (1.1 g, 8.2 mmol),
(BOC)2O (2.76 g, 12.3 mmol) and DMAP (100 mg) in CH2Cl2
was stirred under N2 at room temperature for 3 hours to give
enantiomerically pure N--BOC lactam 16a, which was used further without
isolation. To this reaction mixture was added 20 ml of 25% aqueous
ammonium hydroxide and stirring was continued for another 4 hours. Water
was added and the reaction mixture was extracted with dichloromethane
(2×50 ml). The combined organic phase was washed with aqueous HCl
(5%), dried over sodium sulfate and reduced to dryness under reduced
pressure to give enantiomerically pure N--BOC carboxyamide 28a (2.51 g)
as a white solid, which was used in the next step without further
purification.

[0191]Procedure: The enantiomerically pure N--BOC carboxyamide 28a (2.51
g) was dissolved in 10 ml dichloromethane and treated with 10 ml TFA. The
mixture was stirred for 1 hour at room temperature and concentrated to
dryness under reduced pressure. The residue was suspended in toluene and
again concentrated to dryness. The resulting solid was dissolved in
methanol:water (30 ml:3 ml) and treated with 1.5 g sodium bicarbonate.
The 5-fluoro-2,4-dichloropyrimidine 34 (3 g, 17.9 mmol) was added and the
mixture was stirred for 2 days at room temperature. The volatiles were
removed under vacuum and the residue was suspended in brine. The
precipitate was filtered, dried and subjected to column chromatography
(silica gel, dichloromethane-methanol, 20:1) to give the desired
enantiomerically pure mono-SNAr product 36a as a white solid (1.7 g,
74%).

[0193]Procedure: A homogeneous mixture of aniline 7 (400 mg, 1.95 mmol),
enantiomerically pure mono-SNAr product 36a (400 mg, 1.41 mmol) and 0.2
ml TFA in 4 ml isopropanol in a sealed tube was stirred at 100° C.
for 20 hours. The mixture was cooled to room temperature, diluted with 2
ml diethylether and the resulting precipitate was filtered and washed
with diethylether. The remaining solids were dissolved in water and
treated with aqueous 25% ammonium hydroxide solution. The resulting
precipitate was filtered, washed with water and dried to give 527 mg
(83%) of desired product, 2,4-pyrimidindiamine derivative 60a as an
off-white solid. Purity was determined by LCMS to be greater than 97% and
the enantiomeric purity was determined by chiral HPLC to be greater than
99%. The chiral analytical data, 1H NMR and LCMS analyses were
identical with the enantiomer that was designated E1.

7.8 Preparation of Stereoisomerically Pure Compounds Using (R)-Methyl-p.
Methoxybenzylamine as a Chiral Auxiliary

[0197]Procedure: A heterogeneous mixture of diasterisomers 20a and 20b
(6.0 g g, 17 mmol), TFA (20 mL) in CH2Cl2 was stirred at room
temperature for 2 hours. TLC was used to monitor the progress of the
reaction. The resulting reaction was concentrated to dryness and dried
under a high vacuum for several hours to afford a diasterisomeric mixture
of intermediates 22a and 22b. This mixture was then reacted with
2,4-dichloro-5-fluoropyrimidine 34 (3.4 g, 20 mmol) in the presence of
NaHCO3 (5.7 g, 68 mmol) in MeOH:H2O (50 mL, each) at room
temperature for 24 hours. The reaction mixture was then diluted with
NaCl-saturated water (50 mL) and extracted with CH2Cl2. The
extract upon drying over anhydrous Na2SO4 followed by removal
of solvent under reduced pressure gave a residue, which was
chromatographed (silica gel, CH2Cl2 then 2% 2N NH3/MeOH in
CH2Cl2). The chromatographic purification gave a mixture
diasterisomers 38a and 38b (4.0 g) (1:1 ratio can be seen with a clear
separation on reverse phase LCMS). The resulting 4.0 grams upon
crystallization using EtOAc:hexanes (30:150 mL; v/v) afforded crystalline
material of intermediate 38a, which was confirmed by X-ray crystal
structure; chemical purity: 96% and % de: 96%. [α]D
-36.7° (c, 0.18 MeOH). The mother liquor containing the other
isomer had poor % de (70-80%), which is assumed to be diastereoisomer
38b.

7.8.3 Preparation of Stereoisomerically Pure Product Including the Chiral
Auxiliary

[0200]The cleavage of chiral auxiliary from 40a was found to be difficult,
therefore the cleavage of chiral auxiliary from intermediate compounds
38a and 38b followed by the second SNAr reaction with aniline 7 was
carried as follows.

[0202]Procedure: The mono-SNAr product with chiral auxiliary 38a was
allowed to react with DDQ (3 equivalents) in CH2Cl2:H2O at
room temperature to obtain the desired mono-SNAr product 36a. The
mono-SNAr product was purified by column chromatography and found to be
same as compound 36a obtained via enzymatic route, which was confirmed by
chiral analytical HPLC, LCMS and 1H NMR. Further, the reaction of
mono-SNAr product 36a with aniline 7 in MeOH:TFA at 100° C. in a
sealed tube for 24 h gave the desired product 60a. It was purified by
column chromatography and analyzed by 1HNMR, LCMS and chiral
analytical HPLC. The chiral analytical HPLC, LCMS and 1H NMR
analyses indicated that the data for the product 60a was matching with
the enantiomer designated E1.

7.8.6 Cleavage of the Chiral Auxiliary from Intermediate 38b and
Preparation of Stereoisomerically Pure
(1S,2S,3R,4R)--N-4-(3-Aminocarbonylbicyclo[2.2.1]hept-5-en-2-yl)-5-fluoro-
-N2-[3-methyl-4-(4-methylpiperazin-1-yl)phenyl]-2,4-pyrimidinediamine

[0203]Reaction:

##STR00071##

[0204]Procedure: The mono-SNAr product 38b was allowed to react with DDQ
(3 equivalents) in CH2Cl2:H2O at room temperature to
obtain the desired mono-SNAr product 36b (after the cleavage of chiral
auxiliary). The mono-SNAr product was purified by column chromatography
and found to be a different diastereoisomer than that was obtained via
enzymatic route, and this was confirmed by chiral analytical HPLC.
Further, the reaction of mono-SNAr product 36b with aniline 7 in MeOH:TFA
at 100° C. in a sealed tube for 24 h gave the desired product 60b.
It was purified by column chromatography and analyzed by 1HNMR, LCMS
and chiral analytical HPLC. The chiral analytical HPLC, LCMS and 1H
NMR analyses indicated that the data for product 60b was identical with
the enantiomer designed E2. [α]DRT -102.00° (c,
1.0 MeOH).

7.9 Preparation of HCl Salts

[0205]HCl salts of the racemate 60r1 and stereoisomerically pure 60a were
prepared as described below.

[0211]The IC50 values obtained with the compounds are provided in
TABLE 2, below. In TABLE 2, a "+" indicates an IC50 value of
≦1 μM, a "++" indicates an IC50 value of ≦20 nM,
"+++" indicates an IC50 value of ≦10 nM, and a "-" indicates
an IC50 value of >1 μM. A blank indicates that the compound
was not tested against the specific cell line.

[0212]Compounds 60a and 60b were tested for their ability to inhibit
Aurora kinase-B in a functional cellular assay involving phosphorylation
of its substrate, histone H3. For the assay, A549 cells were seeded into
the wells of a microtiter tray (5000 cells/well in 100 μl F12K media)
late in the afternoon on Day 1. The cells were grown overnight
(37° C., 5% CO2). On Day 2, 50 μl nocodazole (1 μM in
media) was added to each well, giving a final concentration of 333 nM.
Cells were grown for an additional 18 hrs under the same conditions.

[0214]A Zeiss Axiovert S100 inverted fluorescent microscope with a
Plan-NEOFLUAR 10× objective, a Hamamatsu Lightningcure 200
Mercury-Xenon light source and an Omega Optical XF57 quad filter was used
for all data collection. The system was equipped with a Ludl Mac2000
motorized stage with X/Y/Z control, a Ludl filter wheel, a Zymark Twister
robot arm and a Quantix digital camera from Roper Scientific. All
hardware was controlled with ImagePro 4.5 with the ScopePro/StagePro 4.1
module (Media Cybernetics) on a PC running Win2000. Visual Basic Scripts
were written for ImagePro to automate hardware control and image
collection. Focusing was performed with a software auto-focus routine
contained with StagePro that used the maximum local contrast to determine
the best plane of focus from a Z series captured once in each well. Once
proper focus was achieved images were captured in a 3×3 grid
pattern of adjacent images next to, but not including, the position of
focusing. Images were captured and analyzed in 12-bit format using
segmentation and morphological routines contained in the Image Pro
software package. Identified nuclei were counted and pixel data for each
cell along with experimental conditions was stored in a database using
MySQL 4.0.14. Subsequent analysis of experimental results and graph
creation was done using Matlab 6.5.

[0215]For phospho-histone H3 analysis the data is converted to Facs files
and analysed using FlowJo. The percent Phospho-H3 cells are plotted at
each compound concentration to determine an EC50 for Aurora B inhibition.

[0216]Results. Compound 60a inhibited Aurora kinase-B with an IC50 of
about 7 nM in this assay. By contrast, the IC50 of its enantiomer,
compound 60b, was 2.49 μM, approx. 350 times greater.

7.13 Pharmacokinetics of Compound E1 in Monkeys

[0217]Compound 60a was administered to monkeys intravenously (1 mg/kg in
saline) and orally (5 mg/kg in saline) and the plasma concentrations
monitored over time. When administered by i.v., the plasma concentration
of compound remained above the IC50 of 7 nM for 11 hrs following
administration; when administered orally, a plasma concentration of
compound above the IC50 was maintained for over 20 hrs.

7.14 Compound 60a Shrinks Tumors In Vivo

[0218]Compound 60a.2HCl, was tested for its ability to shrink A549 and
Colo205 tumors in a standard xenograft therapeutic model in SCID mice,
and Colo205 and MiaPaCa tumors in a standard xenograph regression model
in SCID mice. When palpable tumors appeared and were of a preselected
volume (approx. 100 mm3 for treatment model; >300 mm3 for regression
model), the mice were administered test compounds in the amounts and
according to the dosing regimens specified in TABLE 3 (treatment
protocol) and TABLE 4 (regression protocol), below.

[0219]Results. The inhibitory effects of Compound 60a.2HCl on Colo205
tumor growth in the treatment model are illustrated in FIGS. 1 and 2. The
results of the daily dosing regimen are illustrated in FIG. 1; the
results of the pulsed dosing regimens in FIG. 2. Both dosing regimens
yielded significant (p<0.050) reductions in tumor growth rate as
compared to a vehicle control for all dosage levels tested. A 549 tumors
were less responsive to treatment resulting in an approximate 40%
reduction in mean tumor volume following a dosing regimen of 5 days on/2
days off and a dose level of 10 mg/kg qd (p>0.05).

[0220]The inhibitory effects of Compound 60a.2HCl on Colo205 tumor growth
in the regression model are illustrated in FIG. 3. The effects of
Compound 60a.2HCl on MiaPaCa tumors in the regression model are
illustrated in FIG. 4. Significant reductions in tumor growth rate were
observed with both tumor lines. These reductions were independent of the
mode of administration. Moreover, the reductions observed in MiaPaCa
tumors were similar to those observed with taxol (see FIG. 4).

[0221]Although the foregoing inventions have been described in some detail
to facilitate understanding, it will be apparent that certain changes and
modifications may be practiced within the scope of the appended claims.
Accordingly, the described embodiments are to be considered as
illustrative and not restrictive, and the invention is not to be limited
to the details given herein, but may be modified within the scope and
equivalents of the appended claims.

[0222]All literature and patent references cited throughout the
application are incorporated into the application by reference for all
purposes.